The present invention relates to a novel endogenous compound 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid which is useful for testing a biological function, to an antibody specifically reactive with the novel endogenous compound 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid, to a hybridoma that produces the antibody, to a method for testing a biological function comprising quantitating the novel compound 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid in a biological sample and determining the biological function based on the quantitated value, to a method for immunologically quantitating 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid by using the antibody, and to a process for synthesizing 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
Test of biological functions is very important, for example, for diagnosing diseases or for confirming therapeutic effects. For such test of biological function, various endogenous compound have been used as markers heretofore. Diseases are diagnosed or therapeutic effects are confirmed, based on the presence or concentration of the marker contained in a collected biological sample.
There have been demands for further detailed diagnosis of biological functions such as renal function, central nervous function and developmental function of fetus, and development of novel markers suitable for such diagnosis and development of uses thereof have been strongly demanded.
For example, glomerular filtration rate (hereinafter, abbreviated as xe2x80x9cGFRxe2x80x9d) is one of the best indexes for testing renal functions. Accurate GFR can be measured by inulin clearance method. However, this measurement method is quite hard for the subject to bear, requiring patience and restraint of the subject. An alternative method is a creatinine clearance method which tests GFR based on creatinine levels in blood and creatinine excretion levels in urine. Creatinine can be easily quantitated by chemical or enzymatic methods. However, it is difficult to obtain accurate GFR by the creatinine clearance method since creatinine is secreted from uriniferous tubule; since its in vivo synthesis level is dependent on physiological parameters such as age, sex, body weight, obesity and the like; and since creatinine levels in blood and urine are increased due to intestine absorption of creatinine after ingestion of cooked meat or the like containing creatinine. Therefore, a novel index as an alternative to inulin and creatinine is strongly demanded.
The objects of the present invention are to provide a method for testing biological functions such as renal function, central nervous function and developmental function of fetus by quantitating a level of a novel endogenous compound; to provide a method for immunologically quantitating the novel endogenous compound; and to provide an antibody used therefor. Furthermore, the objects of the invention are to provide a process for producing the antibody used in the method for immunologically quantitating the novel endogenous compound; to provide a novel compound that can be used as an immunogen or as a hapten in the antibody production process; and to provide a process for synthesizing the novel compound. Moreover, the object of the present invention is to provide a novel intermediate for synthesis of the novel compound. The further object of the invention is to provide a hybridoma that produces an antibody to the novel endogenous compound.
The present inventors have analyzed various substances contained in biological samples by high-performance liquid chromatography, and found the existence of a novel fluorescent substance that appears at a high in vivo level in patients suffering from biological dysfunctions, for example, patients with renal dysfunction, pregnant patients with uremic syndrome and patients with central nervous dysfunction, thereby accomplishing the present invention.
The present invention relates to a method for testing a biological function, comprising: quantitating 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid in a collected biological sample; and determining the biological function based on the quantitated value.
Examples of the above-mentioned biological function include a renal function, a central nervous function, and a developmental function of fetus. The biological sample is, for example, urine, serum, cerebrospinal fluid or amniotic fluid.
The present invention also relates to a method for immunologically quantitating 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid, comprising immunologically quantitating 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid in a sample by using an antibody specifically reactive with 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
The present invention further relates to an antibody specifically reactive with 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid. The antibody is, for example, a monoclonal antibody. The monoclonal antibody is, for example, monoclonal antibody KTM-250.
The present invention also relates to a hybridoma which produces a monoclonal antibody specifically reactive with 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid. Examples of the hybridoma include hybridome KTM-250 (FERM BP-6432).
The present invention also relates to a 2-amino-3-[2-(xcex1-D-mannopyranosyl)indole-3-yl]propionic acid derivative represented by general formula (XV): 
wherein R1a to R4a, which may be the same or different, independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group; R5a represents a hydrogen atom, a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; R6a represents a hydrogen atom or a substituted or unsubstituted alkyl group; and R7a represents a hydrogen atom or a substituted or unsubstituted aralkyloxycarbonyl group, or 2-amino-3-[2-(xcex1-L-mannopyranosyl)indole-3-yl]propionic acid derivative represented by general formula (XVxe2x80x2): 
wherein R1a to R4a, which may be the same or different, independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group; R5a represents a hydrogen atom, a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; R6a represents a hydrogen atom or a substituted or unsubstituted alkyl group; and R7a represents a hydrogen atom or a substituted or unsubstituted aralkyloxycarbonyl group. Examples of the derivative include 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
The present invention also relates to a process for producing an antibody to 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid, wherein the process uses, as an immunogen or as a hapten, a 2-amino-3-[2-(xcex1-D-mannopyranosyl)indole-3-yl]propionic acid derivative represented by general formula (XV): 
wherein R1a to R4a, which may be the same or different, independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group; R5a represents a hydrogen atom, a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; R6a represents a hydrogen atom or a substituted or unsubstituted alkyl group; and R7a represents a hydrogen atom or a substituted or unsubstituted aralkyloxycarbonyl group,
or a 2-amino-3-[2-(xcex1-L-mannopyranosyl)indole-3-yl]propionic acid derivative represented by general formula (XVxe2x80x2): 
wherein R1a to R4a, which may be the same or different, independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group; R5a represents a hydrogen atom, a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; R6a represents a hydrogen atom or a substituted or unsubstituted alkyl group; and R7a represents a hydrogen atom or a substituted or unsubstituted aralkyloxycarbonyl groups. The above-described immunogen or hapten is, for example, 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
The present invention also relates to a process for producing a 2-amino-3-[2-(xcex1-D-mannopyranosyl)indole-3-yl]propionic acid derivative represented by general formula (XV): 
wherein R1a to R4a, which may be the same or different, independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group; R5a represents a hydrogen atom, a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; R6a represents a hydrogen atom or a substituted or unsubstituted alkyl group; and R7a represents a hydrogen atom or a substituted or unsubstituted aralkyloxycarbonyl group,
or a 2-amino-3-[2-(xcex1-L-mannopyranosyl)indole-3-yl]propionic acid derivative represented by the following formula (XVxe2x80x2): 
wherein R1a to R4a, which may be the same or different, independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group; R5a represents a hydrogen atom, a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; R6a represents a hydrogen atom or a substituted or unsubstituted alkyl group; and R7a represents a hydrogen atom or a substituted or unsubstituted aralkyloxycarbonyl group,
the process comprising synthesizing a compound represented by general formula (IV) 
wherein R1 to R4, which may be the same or different, independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group,
or a compound represented by general formula (IVxe2x80x2): 
wherein R1 to R4, which may be the same or different, independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group,
by reacting a D-mannose derivative represented by general formula (I) 
wherein R1 to R4, which may be the same or different, independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group,
or a L-mannose derivative represented by general formula (Ixe2x80x2) 
wherein R1 to R4, which may be the same or different, independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group,
with an organometallic reagent represented by general formula (III) 
wherein R5 represents a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group, or a substituted or unsubstituted alkoxymethyl group; and M represents a metal atom, a metal halide, an organometal or a metal salt. An example of the above-described product is 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
Hereinafter, the compound represented by general formula (I) will be referred to as xe2x80x9ccompound (I)xe2x80x9d. Other compounds represented by different formulae will also be expressed likewise.
Physicochemical properties of the novel endogenous compound obtained according to the present invention are as follows:
(1) Color of the substance: colorless
(2) Mass spectrometry
m/z 365 [Mxe2x88x92H]xe2x88x92, m/z 367 [M+H]+
Molecular weight: 366
(3) High resolution mass spectrometry
found: 367.1528 [M+H]+, calculated for C17H23N2O7: 367.1505
(4) Nuclear magnetic resonance spectrum
1H-NMR (500 MHz, D2O) xcex4: 3.35(1H, dd, J=8.8, 15.4 Hz; H-xcex2), 3.55(1H, dd, J=5.1, 15.4 Hz; H-xcex2), 3.74 (1H, dd, J=3.4, 12.7 Hz; H-6xe2x80x2), 3.90 (1H, ddd, J=3.4, 3.4, 9.0 Hz; H-5xe2x80x2), 3.95 (1H, dd, J=3.4, 5.1 Hz; H-4xe2x80x2), 4.02 (1H, dd, J=5.1, 8.8 Hz; H-xcex1), 4.12 (1H, dd, J=3.2, 5.1 Hz; H-3xe2x80x2), 4.25 (1H, dd, J=9.0, 12.7 Hz; H-6xe2x80x2), 4.43 (1H, dd, J=3.2, 8.3 Hz; H-2xe2x80x2), 5.18 (1H, d, J=8.3 Hz; H-1xe2x80x2), 7.22 (1H, dd, J=8.0, 8.0 Hz; H-5), 7.31 (1H, dd, J=8.0, 8.0 Hz; H-6), 7.53 (1H, d, J=8.0 Hz; H-7), 7.74 (1H, d, J=8.0 Hz; H-4).
13C-NMR (125 MHz, D2O) xcex4: 28.4 (C-xcex2), 57.7 (C-xcex1), 61.5 (C-6xe2x80x2), 68.6 (C-1xe2x80x2), 70.1 (C-2xe2x80x2), 71.3 (C-4xe2x80x2), 72.9 (C-3xe2x80x2), 81.4 (C-5xe2x80x2), 110.8 (C-3), 114.3 (C-7), 121.2 (C-4), 122.6 (C-5), 125.3 (C-6), 129.5 (C-3a), 135.8 (C-7a), 138.4 (C-2). 177.1 (COOH).
(5) Main absorption bands at infrared absorption spectrum (KBr method) cmxe2x88x921: 3400-3200, 2932, 1630, 1497, 1460, 1406, 1348, 1243, 1077, 1013, 748.
According to these data, the chemical structure of the novel endogenous compound of the invention was determined to be 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
Hereinafter a process for producing 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid will be described.
The novel endogenous compound of the invention can be obtained from a body fluid such as urine, serum or cerebrospinal fluid from animals through its isolation and purification. The compound may be isolated and purified by routine methods utilizing physicochemical differences such as differences in molecular weights, charges, polarities, hydrophobicities, adsorption coefficients, distribution coefficients, solubilities in various solutions and specific affinities.
Purification is conducted according to known methods using various chromatographies (e.g., column chromatography or thin-layer chromatography). Examples of the chromatography include ion-exchange chromatography, gel-filtration chromatography, adsorption column chromatography, partition column chromatography, hydrophobic chromatography and isoelectric chromatography, which may be used alone or in combination. High-performance liquid column chromatography may also be employed. Procedures involved in the chromatography such as desalting, concentration and extraction may be performed according to conventional methods.
Insoluble substances such as precipitates or high-molecular-weight compounds such as proteins contained in the collected biological sample are preferably removed prior to the chromatography process. The insoluble substances such as precipitates can be removed, for example, by centrifugation or filtration. The high-molecular-weight compounds like proteins can be removed, for example, by denaturation with organic solvents or the like, or by heat treatment.
The novel endogenous compound of the invention can be detected upon the above-described isolation and purification process, for example, by high-performance liquid column chromatography under the following conditions:
Column: Finepak SIL C18-T5 (4 mmxc3x9725 cm, JASCO Corporation)
Development solvent: a mixed solvent of acetonitrile and a buffer (70 mM citric acid: 60 mM disodium phosphate=1:1, v/v, pH 3.5)
0 to 1.5 min.; acetonitrile:buffer=96:4 (v/v)
1.5 to 7.0 min.; acetonitrile:buffer=92:8 (v/v)
7.0 to 25.0 min.; acetonitrile:buffer=88:12 (v/v)
Temperature: room temperature
Flow rate: 1.5 ml/min.
Detection: Fluorescence, excited at 302 nm, determined at 350 nm
Retention time: about 6.8 minutes
Compounds (XIII) and (XIIII) of the present invention may be obtained by synthesis described below. According to the present invention, compounds (XIXX) and (XIIIxe2x80x2) are obtained when the D-mannose derivative represented by formula (I) and L-mannose derivative represented by formula (Ixe2x80x2) are used as starting materials, respectively. Compound (XIII) contains (2R)-2-amino-3-[2-(xcex1-D-mannopyranosyl)indole-3-yl]propionic acid and (2S)-2-amino-3-[2-(xcex1-D-mannopyranosyl)indole-3-yl]propionic acid) while compound (XIIIxe2x80x2) contains (2R)-2-amino-3-[2-(xcex1-L-mannopyranosyl)indole-3-yl]propionic acid and (2S)-2-amino-3-[2-(xcex1-L-mannopyranosyl)indole-3-yl]propionic acid.
A synthesis pathway for producing compound (XIII) from compound (I) via compound (IV) and a synthesis pathway for producing compound (XIIIxe2x80x2) from compound (Ixe2x80x2) via compound (IVxe2x80x2) are shown below, respectively. Since compound (Ixe2x80x2) is an enantiomer of compound (I), compound (XIIIxe2x80x2) may be obtained from compound (Ixe2x80x2) via compound (IVxe2x80x2) through the similar synthesis pathway. Compounds represented by formulae (XV) and (XVxe2x80x2) are obtained by synthesis according to the reaction pathway of this synthesis. 
wherein R1 to R4, which may be the same or different, independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group or a substituted or unsubstituted aryl group; R5 represents a substituted or unsubstituted sulfamoyl group, a substituted or unsubstituted arylsulfonyl group, a substituted or unsubstituted alkoxycarbonyl group or a substituted or unsubstituted alkoxymethyl group; X represents a hydrogen atom or a halogen atom; M represents a metal atom, a metal halide, an organometal or a metal salt; R6 represents a substituted or unsubstituted alkyl group; and Ar represents a substituted or unsubstituted aryl group.
In defining each of the above-mentioned groups, examples of alkyl moieties of the alkyl, alkoxycarbonyl and alkoxymethyl groups include linear or branched C1-6 alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, isoamyl and hexyl groups.
Examples of the alkenyl group include linear or branched C2-6 alkenyl groups such as vinyl, 1-propenyl, 2-propenyl (allyl), butenyl, pentenyl and hexenyl groups.
Aryl moiety of the aryl or aralkyloxycarbonyl group represents a phenyl group, a naphthyl group, or the like. Alkylene moiety of the aralkyloxycarbonyl group represents a moiety formed by removing one hydrogen atom from the above-described alkyl group.
Substituents of the substituted alkyl or alkenyl group, which may be the same or different, include an aryl group, an alkoxy group, an amide group, an amino group, a halogen atom, a carboxyl group, a nitro group, a hydroxy group, a sulfo group and the like. In this case, the number of the substituents may be 1 to 3.
Substituents of the substituted aryl group, substituents on the aryl of the substituted arylsulfonyl group or the substituents on the aryl of the substituted aralkyloxycarbonyl group, which may be the same or different, include an alkyl group, an alkoxy group, an amide group, an amino group, a halogen atom, a carboxyl group, a nitro group, a hydroxy group, a sulfo group and the like. In this case, the number of substituents may be 1 to 3.
Substituents on the nitrogen atom of the substituted sulfamoyl group, which may be the same or different,include a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group and the like. In this case, the number of substituents may be 1 to 2. The substituents of the substituted aryl and alkyl groups are as described above.
Examples of the substituted alkoxycarbonyl group include methoxycarbonyl, tert-butoxycarbonyl and 2,2,2-trichloroethoxycarbonyl groups.
Examples of the substituted alkoxymethyl group include methoxymethyl and 2-trimethylsilylethoxymethyl groups.
The halogen atom refers to a fluorine, chlorine, bromine or iodine atom.
The alkyl moiety of the alkoxy group has the same meanings as the above-described alkyl group.
Preferably, R1 to R4 of compounds (I) and (Ixe2x80x2) independently represent a benzyl group. D-mannose derivative (I) may be synthesized, for example, by the method described in J. Am. Chem. Soc., 104, 4976 (1982). L-mannose derivative (Ixe2x80x2) may be synthesized, for example, from compound (XIV) as described in J. Med. Chem., 29, 1945 (1986) [wherein R1 to R4 independently represent a benzyl group], for example, by the method described in J. Am. Chem. Soc., 104, 4976 (1982).
Step 1
Organometallic reagent (III) can be prepared from compound (II) in the following manner:
(i) Reacting compound (II), wherein X is a hydrogen atom, with an organolithium reagent (proton-metal exchange reaction); (ii) reacting compound (II), wherein X is a halogen atom, with a metal itself or an organometallic reagent (halogen-metal exchange reaction); and (iii) reacting the organometallic reagent prepared in (i) or (ii) with an inorganic metal salt or organometallic compound (metal-metal exchange reaction).
M of organometallic reagent (III) may be a lithium atom, a magnesium salt, a cerium salt, a zinc salt, a copper salt, a mercury salt or the like, preferably a lithium atom.
The organometallic reagent (III), wherein M is a lithium atom, may be prepared from compound (II) and an organolithium reagent. When compound (II), wherein X is a hydrogen atom, is used, the organolithium reagent may be n-butyllithium, lithium diisopropylamide, sec-butyllithium, sec-butyllithium-tetramethylethylenediamine or tert-butyllithium, preferably lithium diisopropylamide. When compound (II), wherein X is a halogen atom, is used, n-butyllithium, sec-butyllithium, sec-butyllithium-tetramethyl ethylenediamine or tert-butyllithium may be used as the organolithium reagent. The organolithium reagent may be used in an amount of 0.7 to 1.0 equivalent, preferably 0.8 to 0.9 equivalent relative to the amount of compound (II).
The reaction temperature is xe2x88x92100xc2x0 C. to xe2x88x9260xc2x0 C., preferably xe2x88x9280xc2x0 C. to xe2x88x9270xc2x0 C. The reaction time varies depending on the organolithium reagents used, and generally is 5 to 30 min.
Step 2 or 2xe2x80x2
Compound (IV) or (IVxe2x80x2) may be obtained by respectively reacting compound (I) or (Ixe2x80x2) with organometallic reagent (III). Organometallic reagent (III) is used in an amount of 1.1 to 3.0 equivalents, preferably 1.2 to 1.4 equivalents relative to the amount of compound (I) or (Ixe2x80x2).
The reaction solvent may be an ether solvent such as diethylether or tetrahydrofuran, or a hydrocarbon solvent such as n-hexane or toluene, the solvents being used alone or in mixture.
The reaction temperature is xe2x88x92100xc2x0 C. to xe2x88x9260xc2x0 C., preferably xe2x88x9280xc2x0 C. to xe2x88x9270xc2x0 C. The reaction time is 10 min. to 2 hours, generally about 30 min.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, termination of the reaction by addition of an aqueous saturated ammonium chloride solution or the like, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 3 or 3xe2x80x2
Compound (V) or (Vxe2x80x2) can be obtained by respectively reacting compound (IV) or (IVxe2x80x2) with a reducing agent. The reducing agent is used in an amount of 3.0 to 10.0 equivalents, preferably 3.0 to 4.0 equivalents relative to the amount of compound (IV) or (IVxe2x80x2).
The reducing agent may be a metal-hydrogen complex compound such as lithium aluminum hydride or sodium borohydride, preferably lithium aluminum hydride.
The reaction solvent, although it varies depending on the bases used, may be an ether solvent such as diethyl ether or tetrahydrofuran, or optionally a protonic solvent such as methanol, the solvents being used alone or in mixture.
The reaction temperature, although it may vary depending on the bases used, may be xe2x88x9240xc2x0 C. to room temperature, generally 0xc2x0 C. to room temperature. The reaction time, although it may vary depending on the reaction temperatures and the bases used, is generally 30 min. to 2 hours.
The reaction may be followed by a series of steps employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, termination of the reaction by addition of an aqueous saturated Rochelle salt solution or the like, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 4 or 4xe2x80x2
Compound (VI) or (VIxe2x80x2) can be obtained by respectively reacting compound (V) or (Vxe2x80x2) with an acid. The acid is used in an amount of 0.1 to 1.0 equivalent, preferably 0.5 to 0.6 equivalent relative to the amount of compound (V) or (Vxe2x80x2).
The acid may be an organic acid such as par-toluene sulfonate or camphor sulfonate, or an inorganic acid such as sulfuric acid or hydrochloric acid, preferably para-toluene sulfonate.
The reaction solvent may be an aromatic hydrocarbon solvent such as toluene or benzene, or a halogenated hydrocarbon solvent such as dichloromethane or chloroform, preferably toluene.
The reaction temperature, although it may vary depending on the acids used, is generally a temperature around the boiling point of the solvent used. The reaction time, although it may vary depending on the acids used, is generally 1 to 2 hours.
The reaction may be followed by a series of steps employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, termination of the reaction by addition of an aqueous saturated sodium hydrogencarbonate solution or the like, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 5 or 5xe2x80x2
Alternatively, compound (VI) or (VIxe2x80x2) can also be obtained by respectively reacting compound (IV) or (IVxe2x80x2) with a reducing agent in the presence of an acid. The acid is used in an amount of 1 to 50 equivalents, preferably 10 to 15 equivalents relative to the amount of compound (IV) or (IVxe2x80x2). The reducing agent is used in an amount of 1.0 to 5.0 equivalents, preferably 1.5 to 2.0 equivalents relative to the amount of the acid.
The acid may be Lewis acid such as boron trifluoride diethyl etherate or zinc chloride, or a protonic acid such as sulfuric acid, acetic acid or trifluoroacetic acid, preferably boron trifluoride diethyl etherate.
The reducing agent may be triethylsilane or the like.
The reaction solvent may be a non-protonic polar solvent such as acetonitrile, an aromatic hydrocarbon solvent such as toluene or benzene, or a halogenated hydrocarbon solvent such as dichloromethane or chloroform.
The reaction temperature, although it may vary depending on the solvents used, is xe2x88x92100xc2x0 C. to a temperature around the boiling point of the solvent used, generally xe2x88x9278xc2x0 C. or xe2x88x9240xc2x0 C. to room temperature. The reaction time is generally 10 to 20 hours.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, termination of the reaction by addition of an aqueous saturated sodium hydrogencarbonate solution or the like, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 6 or 6xe2x80x2
Compound (VII) or (VIIxe2x80x2) can be obtained by respectively reacting compound (VI) or (VIxe2x80x2) with an excessive amount of a base. The base is preferably used in an amount of 50 to 150 equivalents relative to the amount of compound (VI) or (VIxe2x80x2).
The base may be alkali metal hydroxide such as lithium hydroxide, sodium hydroxide and potassium hydroxide, alkali metal alkoxide such as sodium methoxide, sodium ethoxide and potassium tert-butoxide, alkali metal carbonate such as sodium carbonate and potassium carbonate, or the like.
The reaction solvent may be a mixed solvent of water and an alcohol such as methanol, ethanol or isopropanol, or a mixed solvent of water and an ether solvent such as diethylether, tetrahydrofuran or 1,2-dimethoxyethane.
The reaction temperature is not specifically limited, but preferably a temperature around the boiling point of the solvent used. The reaction time, although it varies depending on the bases and solvents used, is generally 1 to 20 hours.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, addition of an aqueous saturated ammonium chloride solution or the like, followed by extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Alternatively, compound (VII) or (VIIxe2x80x2) may also be obtained by respectively reacting compound (VI) or (VIxe2x80x2) with a reducing agent.
The reducing agent may be a metal-hydrogen complex compound such as lithium aluminum hydride or sodium borohydride, or a metal such as magnesium or zinc, the reducing agent being used optionally in the presence of an inorganic salt such as ammonium chloride or an acid such as hydrochloric acid.
The reaction solvent, although it varies depending on the reducing agents used, may be an ether solvent such as diethylether and tetrahydrofuran, or optionally a protonic solvent such as methanol.
The reaction temperature, although it may vary depending on the reducing agents used, is generally room temperature to a temperature around the boiling point of the solvent used. The reaction time, although it differs depending on the reducing agents used, is generally 2 to 24 hours.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, addition of an aqueous saturated ammonium chloride solution or the like, followed by extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 7 or 7xe2x80x2
Compound (IX) or (IXxe2x80x2) can be obtained by respectively reacting compound (VII) or (VIIxe2x80x2) with compound (VIII) in the presence of a base. Compound (VIII) may be synthesized according to the method described in J. Chem. Soc., Chem. Commun., 1089 (1979). Compound (VIII) is used in an amount of 1.0 to 10 equivalents, preferably 2.0 to 2.5 equivalents relative to the amount of compound (VII) or (VIIxe2x80x2), and the base is used in an amount of 1.0 to 10 equivalents, preferably 1.5 to 2.0 equivalents relative to the amount of compound (VIII).
Examples of the base include alkali metal carbonate such as sodium carbonate and potassium carbonate.
The reaction solvent may be a halogenated hydrocarbon solvent such as dichloromethane, chloroform or 1,2-dichloroethane.
The reaction temperature is, but not limited to, 0xc2x0 C. to a temperature around the boiling point of the solvent, preferably a temperature around room temperature. The reaction time, although it varies depending on the reaction solvents and bases used, is generally 1 to 2.5 hours.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, addition of an aqueous saturated ammonium chloride solution or the like, followed by extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 8 or 8xe2x80x2
Compound (X) or (Xxe2x80x2) can be obtained by respectively reacting compound (IX) or (IXxe2x80x2) with a reducing agent.
The reducing agent is preferably an amalgam such as aluminum-amalgam or the like. The reaction may be a catalytic reduction using a heterogeneous catalyst such as palladium-carbon, palladium hydroxide-carbon or platinum oxide under an atmosphere of hydrogen gas.
The reaction solvent may be a mixed solvent of water and an ether solvent such as diethyl ether, tetrahydrofuran or 1,2-dimethoxyethane.
The reaction temperature is, but not limited to, 0xc2x0 C. to a temperature around the boiling point of the solvent used, preferably room temperature to 60xc2x0 C. The reaction time, although it varies depending on the solvents used, is generally 0.5 to 1.0 hour.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, filtration of the reaction mixture with celite or the like, addition of an aqueous saturated sodium hydrogencarbonate solution or the like to the filtrate, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 9 or 9xe2x80x2
Compound (XI) or (XIxe2x80x2) can be obtained by respectively reacting compound (X) or (Xxe2x80x2) with arylmethyl chloroformate in the presence of a base. The arylmethyl chloroformate is used in an amount of 1.0 to 10 equivalents, preferably 2.0 to 2.5 equivalents relative to the amount of compound (X) or (Xxe2x80x2). The base is used in an amount of 1.0 to 10 equivalents, preferably 2.0 to 3.0 equivalents relative to the amount of compound (X) or (Xxe2x80x2).
Examples of the base include organic bases such as triethylamine and pyridine, preferably triethylamine.
Examples of the solvent include halogenated hydrocarbon solvents such as dichloromethane, chloroform and 1,2dichloroethane, and aromatic hydrocarbon solvents such as benzene and toluene, preferably chloroform.
The reaction temperature, although it is not specifically limited, is preferably xe2x88x92100xc2x0 C. to room temperature, more preferably xe2x88x9220xc2x0 C. to room temperature. The reaction time is generally 10 min. to 1.0 hour.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, addition of an aqueous saturated sodium hydrogencarbonate solution or the like, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 10 or 10xe2x80x2
Compound (XII) or (XIIxe2x80x2) can be obtained by hydrolyzing compound (XI) or (XIxe2x80x2) with a base, respectively. The base is used in an amount of 1.0 to 10 equivalents, preferably 3.0 to 4.0 equivalents relative to the amount of compound (XI) or (XIxe2x80x2).
Examples of the base include alkyl metal hydroxides such as lithium hydroxide, sodium hydroxide and potassium hydroxide.
The reaction solvent may be a mixed solvent of water and an ether solvent such as diethyl ether, tetrahydrofuran or 1,2-dimethoxyethane, or a mixed solvent of water and an alcoholic solvent such as methanol or ethanol.
The reaction temperature, although it is not specifically limited, is preferably a temperature around room temperature. The reaction time is generally 10 to 24 hours.
The reaction may be followed by a series of steps generally employed in organic synthesis reactions, i.e., termination of the reaction, phase separation, extraction and concentration: for example, addition of an aqueous citric acid solution or the like, extraction with an organic solvent such as ether, ethyl acetate or dichloromethane, drying over anhydrous sodium sulfate or anhydrous magnesium sulfate, and removal of the solvent with a vacuum rotatory evaporator. The compound may be separated/purified by column chromatography, preparative thin-layer chromatography or the like.
Step 11 or 11xe2x80x2
Compound (XIII) or (XIIIxe2x80x2) can be obtained by respectively subjecting compound (XII) or (XIIxe2x80x2) to a hydrogenation using a catalyst under an atmosphere of hydrogen gas.
The catalyst may be a heterogeneous catalyst such as palladium-carbon, palladium hydroxide-carbon or platinum oxide, preferably palladium hydroxide-carbon.
The reaction solvent may be a protonic solvent such as methanol, ethanol or isopropanol, or a non-protonic solvent such as ethyl acetate or N,N-dimethylformamide, the solvents being used alone or in combination. Preferably, the reaction solvent is ethanol.
The reaction temperature is not specifically limited, but it is preferably at room temperature to a temperature around the boiling point of the solvent used, more preferably room temperature to 60xc2x0 C. The reaction time is generally 10 to 15 hours.
The reaction may be followed by operations generally employed in hydrogenation: for example, filtration of the reaction mixture with celite or the like, followed by removal of the solvent with a vacuum rotation evaporator. The synthesized compound may be separated/purified by high-performance liquid column chromatography or the like.
The intermediates obtained by the above steps may directly be used in the subsequent reactions without purification. In the synthesis of the invention, compound (XIII) or (XIIIxe2x80x2) may be obtained in the pure form by isolating/purifying the compound obtained in step 11 or 11xe2x80x2, or preferably by separating/purifying compound (XII) or (XIIxe2x80x2) obtained in step 10 or 10xe2x80x2 by column chromatography, preparative thin-layer chromatography or the like followed by subjecting to the reaction of step 11 or 11xe2x80x2.
Hereinafter, an antibody that is specifically reactive with 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid of the invention will be described.
A process for producing the antibody of the invention is as follows. An antibody to 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid may be produced by using compound (XV) or (XVxe2x80x2) as an immunogen or a hapten. When compound (XV) or (XVxe2x80x2) is used as a hapten, a complex obtained by binding compound (XV) or (XVxe2x80x2) to a high-molecular-weight carrier can be used as an immunogen. Compound (XV) or (XVxe2x80x2) may be used which is obtained by purifying the compound obtained according to the synthetic method described above. Specifically, 2-amino-3-[2-(xcex1-mannnopyranosyl)indole-3-yl]propionic acid may be purified from a biological sample according to the method described above or may be synthesized according to the synthetic method described above.
The high-molecular-weight carrier may be any substance as long as it has a reactive group that can effect a condensation reaction with the carboxyl group, amino group, hydroxy group or the like of compound (XV) or (XVxe2x80x2) and 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid, and as long as it can confer immunogenicity to the compound or it enhances the originally-existing immunogenicity of the compound by linking with the novel tryptophan derivatives. Particular examples of the carrier are bovine serum albumin (hereinafter, abbreviated as xe2x80x9cBSAxe2x80x9d), protein such as globulin, keyhole limpet hemocyanine (hereinafter, abbreviated as xe2x80x9cKLHxe2x80x9d) or thyroglobulin, polysaccharides such as dextran or sepharose, latex particules such as polystylene or acryls, polynucleic acids such as polyuridylic acid or polyalanylic acid, and synthetic polymer such as MAP. These high-molecular-weight substances may be linked with the tryptophan derivatives of the invention according to the methods described by Nobuo Sakado [Procedures for Immunological Experiments 151, Shunsuke Migita et al. (ed.), Nankodo (1995)] such as methods using amino group (e.g., carbodiimide method, glutaraldehyde method, and diisocyanate method), methods using carboxyl group (e.g., activated ester method, mixed anhydride method, and acyl azide method), methods using SH group (e.g., MBS and SPDP methods), and methods using hydroxy group (e.g., cyanogen bromide method and periodate oxidation method).
Examples of animals that may be immunized with the immunogen obtained above include mouse, rat, hamster, rabbit, guinea pig, goat, sheep and chicken. In preparation of polyclonal antibodies the preferred animals are rabbit, guinea pig, goat, sheep and chicken while mouse and rat in preparation of a monoclonal antibodies.
Immunization may be conducted according to the method described by Nishimichi and Toyoshima (New Biochemical Experiments, 1, 389 (1990), Tokyo Kagaku Dojin). For example, the immunogen is emulsified in Freund""s complete or incomplete adjuvant, and administered intraperitoneally, subcutaneously or intramuscularly. For example, immunization may be completed by administering the immunogen twice or more, preferably 2 to 4 times, constantly at 7-30 day intervals, preferably 12-16 day intervals. In obtaining a polyclonal antibody, blood is taken from the completely immunized animal by periodic bleeding or by bleeding of the whole blood. Generally, blood is collected without prevention of coagulation, and is allowed to coagulate prior to recovery of serum fraction by centrifugation or the like. The antibody contained in the blood may be used, if necessary, after purification. Purification may be conducted by various methods including salting-out fractionation using ammonium sulfate or the like, ion exchange chromatography, gel filtration column chromatography, affinity column chromatography using protein A or G, or affinity column chromatography using antigen-immobilized gel, the methods being used alone or in combination.
In obtaining a monoclonal antibody, the antibody-producing cells may be collected from sources such as spleen, lymph node or peripheral blood of immunized animals. Alternatively, the antibody-producing cells may also be obtained by the so-called in vitro immunization [Arai and Ohta, Experimental Medicine, 6:43 (1988)] which comprises removing an antibody-producing competent cell from the spleen, lymph node or peripheral blood of a non-immunized animal, then subjecting the competent cell to a direct immunization.
Myeloma cells fused with the antibody-producing cells are not specifically limited, but preferably are cell lines derived from the same animal as the animal from which the antibody-producing cells are derived. In order to efficiently select only the cells that succeeded in cell fusion, the myeloma cells preferably contain a specific drug marker. For example, a myeloma cell resistant to 8-azaguanine is favored for although it cannot grow in a medium containing hypoxanthine, aminopterin and thymidine (hereinafter, referred to as xe2x80x9cHAT mediumxe2x80x9d), whereas it can grow in HAT medium when it is fused with a normal cell, drawing a distinction between fused and unfused myeloma cells. Specific examples of myeloma cells include P3xc3x9763xe2x88x92Ag.8.653, P3xc3x9763xe2x88x92Ag.8.U1 (hereinafter, simply referred to as xe2x80x9cP3U1xe2x80x9d) and Sp/O-Ag14.
Cell fusion was first developed by Kohler and Milstein [Nature, 256, 495 (1975)], which then rapidly diffused, and various improved methods thereof are currently applicable. According to the common method, the antibody-producing cell and the myeloma cell are mixed in a proportion of 10-3:1 while using 30-50% polyethylene glycol (average molecular weight of 1,500-6,000) as a fusing agent. Their cells may also be fused by an electric pulse [Kawauchi et al., Experimental Medicine, 6, 50 (1988)].
At the end of cell fusion, the cells are suspended in the selective medium and only the fusion cells are grown in a culture container (e.g., a 96-well plate) advantageous for the subsequent selection of the cell of interest. After selectively growing the fusion cells, cells that produce an antibody to the compound of the present invention are selected. This selection is conducted by determining the presence or absence of the antibody of interest contained in the supernatant of the fusion cell culture by a method such as enzyme immunoassay or radioimmunoassay. The selected cells are subjected to, for example, limiting dilution method or soft agar culture method to obtain monoclones, thereby establishing a monoclonal-antibody-producing hybridoma cell line specific for 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid.
The monoclonal antibody may be obtained by culturing an established cell line in a suitable medium and collecting the antibody from the culture. Alternatively, the cell line may be transplanted into the abdominal cavity of an animal and grown in ascites in order to collect the ascites and to recover the monoclonal antibody therefrom. The antibody in the culture or ascites may be, if necessary, purified prior to use. Purification may be conducted by various methods including salting-out fractionation using ammonium sulfate or the like, ion exchange chromatography, gel filtration column chromatography, affinity column chromatography using protein A or G, or affinity column chromatography using antigen-immobilized gel, the methods being used alone or in combination. The antibody can be produced in the above-described manners.
An example of the hybridoma that produces the monoclonal antibody of the invention is hybridoma KTM-250. This hybridoma has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, MITI (Higashi 1-1-3, Tsukuba-shi, Ibaraki-ken, 305-0046, Japan) on Jul. 17, 1998 as FERM BP-6432. Hereinafter, the monoclonal antibody produced by hybridoma KTM-250 is simply referred to as KTM-250 antibody.
Hereinafter, a method for quantitating 2-amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]propionic acid in a biological sample, for use in the test method according to the invention, will be described. The substance in the biological sample may be quantitated by high-performance liquid chromatography using the substance isolated/purified according to the method described above as a standard substance. The measurement instrument may be a general high-performance liquid chromatography instrument which may be equipped, for example, with a liquid transporting pump, a controller, a solvent mixer, a sample injector, a detector and a recorder. The liquid transporting system is desirebly a device that allows concentration-gradient elution. The column is a reverse-phase column or an ion-exchange column. Column filler is preferably porous silica, porous polymer or the like. The substituent of the filler is, for example, C18 alkyl group, C8 alkyl group, phenyl group, diphenyl group, cyanopropyl group, carboxymethyl group, sulfopropyl group, diethylaminoethyl group, or diethyl-(2-hydroxypropyl)aminomethyl group. The detection is conducted by a general detection such as fluorescent detection, absorbance detection, differential refraction detection or electrochemical detection.
If the collected biological sample is measured by high-performance liquid chromatography, the biological sample may directly be injected into the sample injector, or the biological sample may be subjected to pre-treatment. For example, the biological sample may be subjected to centrifugation and its supernatent may be used, or it may be used after filtration through a filter with a pore size of 10 xcexcm or less, preferably 1 xcexcm or less, more preferably 0.1 xcexcm or less. A sample treated in a treatment column may also be used. The biological sample may be subjected to a general steps such as drying, redissolution, extraction, desalting and concentration.
Since the collected biological sample may be unstable, it is preferably added with a reducing agent such as vitamin C, or acid such as hydrochloric acid or perchloric acid.
A fluorescent substance such as N-methylserotonin may be added to the sample as the internal standard for the high-performance liquid chromatography. The solvent used in the high-performance liquid chromatography may be an organic solvent such as acetonitrile, an aqueous sovlent such as trifluoroacetic acid or heptafluorobutyric acid, or a buffer such as phosphate buffer, methane sulfonate buffer, formate buffer, acetate buffer or citrate buffer, which may be used alone or in combination.
Hereinafter, a quantitation method using the antibody will be described. The measurement employed in the present invention will be described.
2-Amino-3-[2-(xcex1-mannopyranosyl)indole-3-yl]proiponate can immunologically be measured by using the antibody of the invention. Examples of the immunological measurement include, but are not limited to, various high-sensitivity immunoassays such as radioimmunoassay using a radioisotope as a label, enzyme immunoassay using an enzyme, fluorescent immunoassay using a fluorescent substance, and luminescent immunoassay using a luminescent substance. Although most of known quantitations are applicable, competitive assays are most suitable. Competitive assays can be carried out in various manners. For example, a labeled antigen may compete with an antigen contained in the sample or with an antigen as a standard for binding to the antibody; an antigen from the sample or an antigen as a standard in the liquid phase may compete with an immobilized antigen for binding to the labeled antibody; or a labeled antigen may compete with an antigen in the sample or an antigen as a standard for binding to the immobilized antibody.
Hereinafter, a method for testing various biological functions will be described. Biological functions are determined from values obtained by quantitating 2-amino-3-[2-(xcex1-mannopyrasyl)indole-3-yl]propionic acid levels in biological samples.
Examples of the biological sample include biological fluids such as blood, plasma, serum, urine, amniotic fluid, cerebrospinal fluid or cell extract, which are selected depending on purposes of the test.
The method of the present invention can be used for assaying an endogenous renal Glomerular filtration rate (hereinafter referred to as GFR).
GFR can be generally expressed as GFR=Uxc3x97V/P (U=a level in urine; V=urine volume; P=a level in blood). Assay of GFR is performed by using as the indication a substance which can freely pass through the renal glomeruli without being degenerated (e.g., decomposed) in vivo and is neither excreted from the uriniferous tubule nor reabsorbed. Since the level of the substance in the glomerular filtrate is equal to that in blood (plasma), the amount of the substance which is excreted in urine during a certain period of time (1 minute) (Uxc3x97V) is equal to that which is filtered through the glomeruli during the same period of time. Accordingly, this amount divided by the level in blood gives a glomerular filtration rate. Generally, test substances include endogenous substances such as creatinine and urea and exogenous substances such as inuline, sodium thiosulfate, and mannitol. Clearance values of these substances are used as GFRs. Since the urinary amount of a person is approximately proportionate to his/her body surface area, such GFR values may be corrected with the body surface area for clinical use. For calculation of urine volume, a value obtained by dividing the amount of a second complete micturition at a certain period of time (1 hour) after a first complete micturition by the time (i.e., 1 hour) (ml/minute) is used. For correction with the body surface area, a value obtained dividing an average body surface area of Japanese people (1.48 m2 for adults) by the body surface area of a subject calculated from his/her height and weight may be used. For example, when creatinine is used, GFR is (65.0xc3x971.44xc3x971.48)/(0.9xc3x971.40)=110 ml/min under the following conditions: urine volume, 1.44 ml/min; creatinine level in urine, 65.0 mg/dl; creatinine level in plasma, 0.9 mg/dl; and body surface area, 1.4 m2.
If, among the substances above, an endogenous substance is produced in vivo at a certain level but exists in a certain level in blood (e.g., renal functions are in an abnormal state such that the endogenous substance cannot be filtered in the kidney), the endogenous substance accumulates in blood and thus its level increases in blood. Accordingly, the disorder of renal functions can be predicted by determining the level in blood and the amount in urine of the endogenous substance. Alternatively, if, regardless of normal renal functions, the level of an endogenous substance is increased in blood or urine, this abnormality may indicate any change in functions such as increased production and decreased metabolism of the substance. In this case, such indicators can be used for diagnosis of disorders. On the contrary, decreased levels of an endogenous substance in blood or urine indicate any change in functions such as decreased production and increased metabolism of the substance, thus such indicators can also be used for diagnostic of disorders. For these purposes, levels of an endogenous substance in blood and in urine (calculated from its level in urine and a urinary amount) may be determined alone or separately.
For testing renal functions, levels of such substances in, for example, serum are determined. Results of Example 3 below show that levels of the substance in sera from 99% of healthy persons (n=50) are within the range of 52-99 ng/ml while the levels in sera from 99% of patients (n=30) with renal dysfunction are within the range of 307-1403 ng/ml. Accordingly, for example, the level of the substance in serum of 100 ng/ml or more indicates a renal hypofunction.
For testing central nervous functions, the level of the substance in, for example, cerebrospinal fluid is determined. Results of Example 4 below show that levels of the substance in cerebrospinal fluids from 99% of healthy persons (n=22) are within the range of 72-188 ng/ml while the levels in cerebrospinal fluids from patients with spinocerebellar ataxia or metastatic brain tumor are 400 ng/ml or more. Accordingly, for example, the level of the substance in cerebrospinal fluid of 200 ng/ml or more indicates a central nervous hypofunction.
For test of pregnancy functions, the level of the substance in, for example, amniotic fluids is determined. Results of Example 5 below show that levels of the substance in amniotic fluids from 99% of healthy persons (n=21) are within the range of 113-593 ng/ml while the levels in amniotic fluids from 99% of pregnant patients with uremic syndrome (n=9) are in the range of 675-2147 ng/ml. Accordingly, for example the level of the substance in amniotic fluids of 600 ng/ml or more indicates pregnant hypofunction.